The endocannabinoids (ECs) A/-arachidonoyl ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are lipid transmitters that activate the central and peripheral cannabinoid receptors (CB1 and CB2, respectively) to regulate a broad range of physiological processes, including pain sensation, inflammation, cognition, emotional state, and feeding. The magnitude and duration of EC signaling are tightly regulated by hydrolytic enzymes. However, our understanding of the distinct biochemical pathways that terminate AEA and 2-AG signaling in vivo remains incomplete. Key outstanding questions include: 1) what are the mechanisms and three-dimensional structures of EC hydrolases? 2) what are the neurochemical and physiological consequences of perturbing the function of EC hydrolases in vivo? 3) do multiple hydrolases coordinately control 2-AG metabolism in vivo? and 4) do AEA- and 2-AG-dependent EC pathways regulate distinct mammalian behaviors? In this Program Project, we have assembled a multidisciplinary research team that aims to address these questions by developing and implementing cutting-edge chemical, enzymological, genetic, proteomic, structural, and behavioral pharmacology methods. Specifically, we plan to: 1) characterize the biochemical and cellular mechanisms for terminating EC signaling the nervous system (Project 1), 2) determine the crystal structures of key EC hydrolases, including apo-enzymes, inhibitor complexes, and active-site mutants (Project 2), and 3) evaluate the neurochemical and behavioral effects of disrupting EC hydrolases in vivo (Projects 3 and 4). The knowledge gained from these studies will be further applied towards the design of increasingly potent and selective inhibitors of EC hydrolases (Core), which should prove of great value as both research tools and potential therapeutic agents for the treatment of a range of human diseases, including chronic pain, depression, anxiety, and metabolic disorders. PUBLIC HEALTH RELEVANCE: The endogenous cannabinoid system regulates a broad range of neurophysiological processes. Elucidation of the enzymes that regulate endogenous cannabinoids and their mechanisms of action may lead to the identification of new therapeutic targets for the treatment of human disorders such as chronic pain, depression, and anxiety.